The object of this proposal is to study the mechanism of Messenger RNA decay. The lac (lactose) operon of Escherichia coli consists of three genes, lacZ, Y and A which encode the proteins beta-galactosidase, lactose permease and thiogalactoside transacetylase. mRNA decay allows the cells to respond to environmental changes rapidly. Thus, the timely decay of mRNA is a very important step in gene regulation. The primary aim of this proposal is to characterize the functional role of a 54 base pair lac Z-Y intergenic space. We intend to show that this space with its stem loop structure can serve as a stabilizer against messenger degradation, thus delays the upstream RNA sequence from decaying. We will use a series of mini-lac operons in which the promoter is fused with different portions of the intergenic space. These plasmids will be tested in vivo for lac specific mRNA. If the stem loop structure protects upstream RNA sequence, then in plasmids containing the complete stem loop structure, the upstream RNA will be preserved. In vitro, we will make RNA transcripts containing the stem loop structure and use them as substrate to test for the effects of several known exonucleases. During lac mRNA decay, a 300 nucleotides fragment is first cleavaged from the 5' end of the lacZ mRNA then chewed back to a 257 nucleotide fragment. We intend to purify a endonuclease that cleavages the 300 nucleotide fragment from the 5' end of the lacZ mRNA. Substrate will be made in vitro using a plasmid, different enzyme preparations will be tested to look for the specific cleavage.